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ORIGINAL ARTICLE  
Year : 2017  |  Volume : 10  |  Issue : 5  |  Page : 1243-1247
A pilot study for the detection of Listeria in cerebrospinal fluid samples from children and adults with signs of meningitis admitted in a tertiary care hospital at Piparia Village, Vadodara, Gujarat, India


Department of Microbiology, SBKS Medical Institute and Research, Sumandeep Vidyapeeth, Piparia, Waghodia, Vadodara, Gujarat, India

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Date of Web Publication6-Nov-2017
 

   Abstract 


Introduction: Human listeriosis (HL) is a foodborne illness causing life-threatening disease of fetus, neonates, elderly, and others with immunosuppression. It is a public health concern because of the severity of disease. Listeria monocytogenes (Lm) has a predilection for central nervous system and placenta. Aims: To culture, cerebrospinal fluid (CSF) samples with special focus on the isolation of Lm. Materials and Methods: A prospective study on CSF samples with special focus on the identification of Lm by direct microscopy (wet film, Gram-stain) and culture on sheep blood agar and a plate of Listeria selective agar (Himedia). Results: 121 CSF samples were processed. Lm was isolated from blood and CSF samples from 8-day-old male neonate admitted with signs of septicemia and meningitis. The isolate was identified using Vitek 2 Compact; Biomerieux. The newborn was fed with goat milk on the 1st day after birth. Conclusions: As against the global focus on HL, documented cases from India are limited. The reasons and difficulties for poor isolation and diagnosis of Lm infections are discussed. Our single patient of neonatal meningitis as a case of community-acquired HL in our area is of significance.

Keywords: Goat milk, Listeria, meningitis, neonate

How to cite this article:
Hemachander S S, Shah KK, Verma Y, Malhotra A, Pandya H, Passi K. A pilot study for the detection of Listeria in cerebrospinal fluid samples from children and adults with signs of meningitis admitted in a tertiary care hospital at Piparia Village, Vadodara, Gujarat, India. Ann Trop Med Public Health 2017;10:1243-7

How to cite this URL:
Hemachander S S, Shah KK, Verma Y, Malhotra A, Pandya H, Passi K. A pilot study for the detection of Listeria in cerebrospinal fluid samples from children and adults with signs of meningitis admitted in a tertiary care hospital at Piparia Village, Vadodara, Gujarat, India. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Dec 15];10:1243-7. Available from: http://www.atmph.org/text.asp?2017/10/5/1243/217510



   Introduction Top


Human listeriosis (HL) is an atypical zoonotic foodborne illness - a life-threatening disease of fetuses, neonates, elderly, and others with immunosuppression. It is a public health concern because of severity of disease with high mortality rate. Listeria monocytogenes (Lm) is ubiquitous and has a predilection for central nervous system and placenta. Diagnosis of listeriosis is rarely done more so, in hospitals of rural areas. Listeriosis is a reportable disease in the USA, France, Israel, etc., Meningitis occurs in many of these cases. Hence, we recruited patients with signs of meningitis in our pilot study for the isolation of Lm, cerebrospinal fluid (CSF) being specimen collected from patients.

Aims and objectives

The aim and objective of this study is to culture CSF samples with special focus on the isolation of Lm.


   Materials and Methods Top


A prospective study on CSF samples was conducted with special focus on isolation of Listeria. Patients admitted to the hospital with the signs of meningitis of all age groups and genders were included in the study. CSF collected in a sterile container aseptically by lumbar puncture was processed immediately. Direct microscopy was done by wet film preparation and Gram-stained smear. Culture: CSF was directly inoculated on sheep blood agar and a plate of Listeria selective agar (Himedia) and incubated at 37°C for 48 h. Identification of bacterial isolate was done by using Vitek 2 GP ID card (Biomeriuex, Missouri, USA).


   Results Top


Of the 121 CSF, samples processed only one yielded positive growth of Lm. The Gram-stained smear from colonies showed tiny Gram-positive cocco bacilli. The isolate was identified by Vitek 2 GP ID card as Lm. [Table 1] shows age and gender distribution of the cases. [Figure 1] shows tiny Gram-positive bacilli of Lm in Gram-stain from the colony. [Figure 2] shows blood agar showing the colonies. Out of 121 samples, 53 were from pediatric age group of which 32 were neonates.
Table 1: Age and gender distribution of patients included in the study and number of the cerebrospinal fluid samples and  Listeria monocytogenes Scientific Name Search nes culture results

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Figure 1: Tiny Gram-positive cocco bacilli of Listeria in Gram-stain smear from the colony

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Figure 2: Blood agar plate showing the colonies of Listeria

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Particulars of the case of listeriosis identified

Male child, 8 days old was admitted to the hospital with a history of fever, convulsions, and refusal of feeds of 1-day duration. Tonic posturing of the limbs, rolling up of the eye balls, 104-degree Fahrenheit temperature was noted. The mother had full-term normal vaginal delivery, baby was healthy at the time of birth, and mother had no history of illness during pregnancy. Noteworthy recording in the case sheet was that the baby was fed on goat milk on the day of birth. The baby developed sickness on the 7th day and was admitted to the hospital on the 8th day. The critically ill neonate (invariably fatal) was discharged from the hospital on the same day against medical advice. The CSF and blood samples were collected immediately after admission for laboratory investigations. The neonate had not received any antibiotics before admission and collection of samples. The isolated strain of Lm was sent for typing. It was identified by PCR as Listeria monocytogenes, serotype 4b.

Lab reports

CSF: Sugar - 20 mg%; protein - 666 mg%; cell count 55 (75% lymphocytes and 25% polymorphs); direct microscopy of CSF showed 0–3 cells and no organisms; culture: Pure growth of Lm from blood and CSF samples.


   Discussion Top


The genus Listeria comprises 10 recognized bacterial species which are widely distributed in our environment but only Lm is a significant human pathogen. Lm is ubiquitous seen in soil, water, sewage, animal and human feces, and even in manures.[1] It possesses the ability to grow in food at high salt concentration, low pH, and low temperature (refrigerated, frozen, packaged, and ready to eat foods). Various contaminated foods such as milk and milk products, meat and meat products, vegetables and seafood, etc., were implicated in major outbreaks and sporadic cases of HL in North America and Europe.[2]

Qi Zhu et al. in the article published in 2017, narrated very well about Lm being found and isolated not only from processed, ready to eat and cold stored meat and dairy products but also in “fresh produce.” Outbreaks of HL associated with fresh produce have been reported in various parts of the world (10 people in Texas in 2010 [chopped celery], 30 people in Colorado with melons, an outbreak in the year 2014 linked to caramel apple contamination in California, form good examples). To quote most recent in January 2016, a multistate outbreak affected 9 states in the USA, packaged salads produced in Ohio being responsible for the outbreak.[3]

Tirumalai had enlisted the reports on the detection of Lm in foods including milk of buffalo, cow, goat, and sheep.[4] Abou-Eleninin et al. in the USA reported that milk samples from 18 of 39 (46.2%) goat farms were positive for Listeria atleast once during their study. Thirty-five samples, i.e. 7.5% of goat milk-yielded Listeria species, Lm identified in 17 out of 35.[5]

Siegman-Igra et al. rightly pointed out in their research paper reviewing 1250 cases of non-perinatal and 494 cases of perinatal HL worldwide, that “ingestion of Lm is a very common occurrence.” However, the development of invasive disease after ingestion of Lm depends on the inoculum size and immune status of the individual. Symptoms vary from person to person. The incubation period (IP) for listeriosis has not been well established.[6] Unlike other foodborne diseases, (IP) for listeriosis can be long, Girard et al. reported an overall median (IP) of invasive listeriosis of 8 days ranging from 1 to 67 days. For pregnancy-associated cases, a longer IP was reported median 27.5 days than for central nervous system (CNS) cases (median 9 days ranging from 1 to 14 days), bacteremia cases (median 2 days, ranging 1–12 days). For febrile gastrointestinal disease, the median IP was reported as 24 h ranging from 6 h to 10 days.[7],[8],[9]

It has been reported that the annual incidence of listeriosis ranges between 1 to 10 per million population.[9] Siegman-Igra et al. in his review of cases worldwide mentioned that average annual incidence was 0.6 for one lakh population between 1995 and 1999.[6] Girard et al. noted annual incidence rate of pregnancy-related listeriosis in France between 1984 and 2011 varied according to the region from 2.2 to 3.6. It was highest in the Paris region and Southwest of France. A total of 3413 cases of listeriosis were recorded from 1999 to 2011, of which 606 (18%) were pregnancy related.[8] Although listeriosis is less common than other foodborne diseases, the foodborne disease-related deaths (19% in the USA, 17% in France) are caused by Lm.[9]

Lm ranked in the top 5 of 31 foodborne pathogens to be the cause of foodborne illnesses by the Centre of Disease Control USA. In the developed countries, listeriosis has emerged as a major foodborne disease. Since it has a high case fatality rate (approximately 20%–30%). About 3.8% of foodborne disease hospitalization and 27.6% of foodborne disease death are due to severe illness caused by Listeria. Since the first documented confirmed foodborne outbreak in 1981, there have been more than 20 listeriosis outbreaks globally as mentioned by Tirumalai.[4] Hence, HL and causative species Lm are being studied globally with increased focus.

In India, Lm is given only little attention. HL case reports in India were enlisted in [Table 1] of Tirumalai article titled listeriosis and Lm in India. This table enlisted 42 HL cases from 1966 to 2011 from India. Out of these, only five were cases of meningitis (North India - 1 (1981): Mumbai - 1 2-month-old male child (1981): Himachal Pradesh - 1 2-month-old male baby (2010), Kashmir - 1 2-month-old female child (2010), South India - 1 (2011)). Other cases are of bad obstetrics history, congenital heart disease, and perinephric abscess, etc.[4] Hence, our single case of an 8-day-old neonate with meningitis is significant because of the rarity and severity of infection caused by Lm. An analysis of 17 Lm isolates recovered from humans in India by multiplex serotyping polymerase chain reaction was given by Kalekar et al. in 2011. The isolates were recovered from clinical samples from patients with various clinical conditions.[10]

Food hygiene and kitchen hygiene will be invariably low in countries where population, poverty and illiteracy coexist, but very limited information is available on the prevalence of HL in India and other developing countries. In the meta-analysis published in Lancet (2014) on global burden of listeriosis, it was clearly stated that the authors were unable to identify the incidence data for AFRO, EMRO, and SEARO WHO regions.[11]

Hospital-acquired listeriosis in adults and among neonates in nurseries reported by Siegman-Igra et al. while reviewing cases worldwide is alarming.[3] A significant feature of the study conducted by Paul et al. in Syndey Australia was detection of 19 cases out of 84 occurring in already hospitalized patients.[12] Hence, after Lm was detected in our laboratory, neonatal intensive care unit was kept under surveillance.

Surveillance of foodborne illness is complicated by several factors. One such factor is underreporting.[13] Many clinical cases go unnoticed and clinical laboratories fail to identify Lm. The reasons for low detection of Lm are to be analyzed. The probable reasons are:

  • Low prevalence of HL
  • Diverse clinical manifestations
  • Lack of awareness about Lm and its severity
  • Atypical nature of Lm
  • Variations in food habits of populations in different geographical areas
  • Nonavailability of rapid/sensitive tests for the laboratory diagnosis or identification of Lm in clinical samples
  • Lack of government regulation in food production, mandatory notification, etc.
  • Errors in identification of Lm in the labs.


Low prevalence of HL

Variation in the prevalence of HL worldwide is variable. Listeriosis is less common than other foodborne diseases.[9]

Diverse clinical manifestations

Listeriosis affects a wide range of patients and results in the diverse array of syndromes as mentioned by Cherubin et al.[14],[15] Nonperinatal, perinatal, and neonatal manifestations of listeriosis are so very varied that physicians can only have a clinical suspicion of the disease. No specific signs and symptoms are present for HL. Moreover, pregnant women may be asymptomatic or present flu-like nonspecific clinical symptoms, backache, headache, vomiting, diarrhea, muscle pains, sore throat, etc.[9]

Lack of awareness about Lm and its severity

As a prelude to our pilot study on Lm, we conducted a small survey by giving questionnaire on listeriosis to a group of 50 medical professionals. Forty people returned the paper unanswered saying that they were not aware of the disease, eight had written that they have heard about it but not aware of the severity of the disease and two clinicians said they never received any laboratory report mentioning Lm during their clinical practice of 12–20 years. They read about Lm in books and journals. This observation of ours is similar to that mentioned by Shrinithivihahshini et al. “In developing and under developed countries, awareness levels of Lm is very low among the physicians.”[16]

Atypical nature of Lm

Listeria is a unique pathogen because it has an intracellular lifecycle. On ingestion of contaminated food, the organism is phagocytosed by the gastrointestinal cells without disrupting the integrity of gastrointestinal tract. Inside the cytoplasm of the cell, it rapidly multiplies and reaches the plasma membrane. Adjacent cells are invaded through plasma membrane protrusions and cell-to-cell spread occurs. By this mechanism, it escapes getting into the extracellular fluid thereby not getting exposed to antibodies, neutrophils, or antibiotics. Cell-mediated immunity is the host-defense against Listeria. Immunocompromised states including pregnancy can predispose to Listeria infection. It can cross the placental barrier and reach the fetus and can also cross the blood-brain barrier and reach the CNS.[7],[9]

Variations in food habits of populations in different geographical areas

Consumption of raw, refrigerated/frozen foods, packaged foods, stored foods and ready to eat foods may be the culprit, as the ubiquitous Lm can survive in these conditions.[4],[13],[16] Pregnant Hispanic women are recognized as having a much higher infection risk than pregnant women of other ethnicities, probably due to the habit of consuming soft cheese![17],[18]

Nonavailability of rapid/sensitive tests for the lab diagnosis or identification of Lm in clinical samples

Tests such as flow cytometry, ELISA, DNA hybridization, PCR, pulse gel electrophoresis, API system, etc., have been developed to specifically detect Listeria species more precisely Lm.[2],[8] Many clinical labs may not have culture facilities.

Lack of government regulation on food production, mandatory notification etc

According to Girard et al., the incidence of pregnancy-related listeriosis is decreased by a factor of 12 from 1984 to 2011 in France. This reduction was the result of progressive implementation of specific Listeria control measures.[8]

Errors in the identification of Lm in labs

Listeria is short Gram-positive cocco bacilli, often confused with diphtheroids (Corynebacterium species), streptococci, etc., It is motile but its motility is due to temperature dependent flagellar expression (tumbling type of motility at 25°C and nonmotile at 37°C). CSF examination in listeriosis shows elevated pressure, increased protein, and increased lymphocyte count.

Error in laboratory reporting can occur for the following reasons:

  • Lm is intracellular, hence, it may be less in numbers in extracellular fluids (body fluids like CSF) until the late stages of the disease
  • Direct smear from CSF may not show sufficient number of cells (polymorphs) to suggest an infection, as there is only increase in lymphocyte count
  • When cells and organisms are less in number in direct smear and presence of growth of Gram-positive bacilli in culture may lead to an error in reporting (may be considered as contaminants such as diphtheroids). Correlation between direct microscopy and growth in culture can go wrong unless there is a clinical suspicion
  • Administration of antibiotics before collection of CSF, blood, etc., from the patient: Culture-negative reporting often occurs in CSF samples (no growth in 90.4% of CSF cultures).[19] Our case of neonatal meningitis yielded pure growth of Lm on routine media, but nothing significant was detected in the direct smear of CSF. The neonate had not received any antibiotics before the collection of samples.


These reasons signify the need for understanding this unique organism called Lm in terms of its biology, behavior, and pathogenesis.


   Conclusions Top


In India, Lm infections have been sporadic. It is not a notifiable disease in India. As against the global focus on HL, documented cases from India are limited. The reasons and difficulties for poor isolation and diagnosis of Lm infections are discussed. Our single patient of neonatal meningitis as a case of community-acquired HL in our area is of significance.

Acknowledgement

We thank the Faculty of Department of Veterinary Microbiology, College of Veterinary Science & Animal Husbandry, Anand, Gujarat, India, for identification of the strain by PCR. Our thanks to Department of Pediatrics, Dhiraj Hospital.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Yadav MM, Roy A, Bhanderi B, Jani RG. Prevalence of Listeria species including L. monocytogenes from apparently healthy animals at Baroda Zoo, Gujarat State, India. J Threatened Taxa 2011;3:1929-35.  Back to cited text no. 1
    
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Zhu Q, Gooneratne R, Hussain MA. Listeria monocytogenes in fresh produce: Outbreaks, prevalence and contamination levels. Foods 2017;6. pii: E21.  Back to cited text no. 3
    
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Tirumalai PS. Listeriosis and Listeria monocytogenes in India. Wudpecker J Food Technol 2013;1:98-103.  Back to cited text no. 4
    
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Abou-Eleinin AA, Ryser ET, Donnelly CW. Incidence and seasonal variation of Listeria species in bulk tank goat's milk. J Food Prot 2000;63:1208-13.  Back to cited text no. 5
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Siegman-Igra Y, Levin R, Weinberger M, Golan Y, Schwartz D, Samra Z, et al. Listeria monocytogenes infection in Israel and review of cases worldwide. Emerg Infect Dis 2002;8:305-10.  Back to cited text no. 6
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Girard D, Leclercq A, Laurent E, Lecuit M, de Valk H, Goulet V, et al. Pregnancy-related listeriosis in France, 1984 to 2011, with a focus on 606 cases from 1999 to 2011. Euro Surveill 2014;19. pii: 20909.  Back to cited text no. 8
    
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Kalekar S, Rodrigues J, D'Costa D, Doijad S, Ashok Kumar J, Malik SV, et al. Genotypic characterization of Listeria monocytogenes isolated from humans in India. Ann Trop Med Parasitol 2011;105:351-8.  Back to cited text no. 10
    
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Paul ML, Dwyer DE, Chow C, Robson J, Chambers I, Eagles G, et al. Listeriosis – A review of eighty-four cases. Med J Aust 1994;160:489-93.  Back to cited text no. 12
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Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999;5:607-25.  Back to cited text no. 13
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Cherubin CE, Appleman MD, Heseltine PN, Khayr W, Stratton CW. Epidemiological spectrum and current treatment of listeriosis. Rev Infect Dis 1991;13:1108-14.  Back to cited text no. 14
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Mylonakis E, Hohmann EL, Calderwood SB. Central nervous system infection with Listeria monocytogenes 33 years' experience at a general hospital and review of 776 episodes from the literature. Medicine (Baltimore) 1998;77:313-36.  Back to cited text no. 15
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Shrinithivihahshini ND, Sheelamary M. Listeriosis, an emerging food-borne disease in India. RRJEES 2016;4:31-3.  Back to cited text no. 16
    
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Pouillot R, Hoelzer K, Jackson KA, Henao OL, Silk BJ. Relative risk of listeriosis in Foodborne Diseases Active Surveillance Network (FoodNet) sites according to age, pregnancy, and ethnicity. Clin Infect Dis 2012;54 Suppl 5:S405-10.  Back to cited text no. 18
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Mengistu A, Gaeseb J, Uaaka G, Ndjavera C, Kambyambya K, Indongo L, et al. Antimicrobial sensitivity patterns of cerebrospinal fluid (CSF) isolates in Namibia: Implications for empirical antibiotic treatment of meningitis. J Pharm Policy Pract 2013;6:4.  Back to cited text no. 19
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Correspondence Address:
S Suguna Hemachander
Department of Microbiology, SBKS Medical Institute and Research, Sumandeep Vidyapeeth, Piparia, Waghodia, Vadodara, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ATMPH.ATMPH_268_17

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